DNA/BSA interaction, bio-activity, molecular docking simulation study and electrochemical properties of hydrazone Schiff base derived Cu(II)/Ni(II) metal complexes: Influence of the nuclearity and metal ions

Article abstract of DOI:10.1016/j.poly.2017.02.047

Three new transition metal complexes of a tridentate Schiff base ligand, H₂L?=?N-(2-hydroxybenzylideneamino) benzamide, were synthesized in the presence of pyridine, 3-methylpyridine and the corresponding metal salts, and were characterized by

Full text of DOI:10.1016/j.poly.2017.02.047

Accepted Manuscript
DNA/BSA interaction, bio-activity, molecular docking simulation study and
electrochemical properties of hydrazone Schiff base derived Cu(II)/Ni(II) metal
complexes: Influence of the nuclearity and metal ions
Roghayeh Fekri, Mehdi Salehi, Asadollah Asadi, Maciej Kubicki
PII:
S0277-5387(17)30188-2
DOI:
http://dx.doi.org/10.1016/j.poly.2017.02.047
POLY 12523
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
7 October 2016
23 February 2017
27 February 2017
Please cite this article as: R. Fekri, M. Salehi, A. Asadi, M. Kubicki, DNA/BSA interaction, bio-activity, molecular
docking simulation study and electrochemical properties of hydrazone Schiff base derived Cu(II)/Ni(II) metal
complexes: Influence of the nuclearity and metal ions, Polyhedron (2017), doi: http://dx.doi.org/10.1016/j.poly.
2
017.02.047
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DNA/BSA interaction, bio-activity, molecular docking simulation
study and electrochemical properties of hydrazone Schiff base
derived Cu(II)/Ni(II) metal complexes: Influence of the nuclearity
and metal ions
a
Roghayeh Fekri , Mehdi Salehi *, Asadollah Asadi , MaciejKubicki
a,
b
c
a
b
Department of Chemistry, College of Science, Semnan University, Semnan, Iran
Department of Biology, Faculty of Science, University of MohagheghArdabili, Ardabil, Iran
c
Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland
Abstract
Three new transition metal complexes of a tridentate Schiff base ligand, H
2
L = N-(2-
hydroxybenzylideneamino) benzamide, were synthesized in the presence of pyridine, 3-
methylpyridine and the corresponding metal salts, and were characterized by FT-IR, UV–Vis
and Raman spectroscopies, and cyclic voltammetry (CV). The crystal structures of the three
II
II
II
complexes, [Cu
2
(L)
2
(py)
2
]·2ClO
4
(1), Cu
2
(L)
2
(3-Mepy)
2
(ClO
4
)
2
(2) and [Ni (L)(py)
3
]ClO
4
(
3), were determined through X-ray crystallography. In the Cu complexes two different kinds
of contacts were be found: shorter (in typical ranges) and longer (Cu-O around 2.5 Å), which
assemble the complexes into dimers. The coordination geometry is (taking into account the
longer contacts) square-pyramidal in 1, while complexes 2 and 3 show an octahedral
geometry. Numerous biological studies have been conducted on H L and complexes 1-3. In
2
vitro, their antibacterial activities were examined against Gram-positive and Gram-negative
bacteria. The results of these studies indicated that all compounds had antibacterial activity
against Gram-positive bacteria. Furthermore, binding studies of H
2
L and its complexes with
L and the
calf thymus DNA (CT-DNA) were studied. Results analyzing the binding of H
2
complexes to CT-DNA revealed a hyperchromic effect and a non-intercalative mode of
binding. These observations also indicate significant alterations of the bovine serum albumin

(
BSA) secondary structure in circular dichroism (CD) spectra, specifically in the presence of
L and the complexes, with the reduction of the α-helices content. A molecular docking
H
2
simulation study of the complexes with Mycobacterium tuberculosis enoyl-acyl carrier
protein reductase, InhA, using the Hex (v8.0.0) docking software indicated their probable
inhibition property.
Keyword: Metal complexes, X-Ray crystal structure, Spectral study, Antibacterial activities,
DNA and BSA binding, Molecular docking simulation.
1
. Introduction
Hydrazone and its analogous compounds have attracted the attention of researchers over the
past two decades, due to their well-known flexible characterization and the capability of
hydrazine to form stable complexes, and they also are considered to be among the most
important stereochemical models [1]. Benzoyl hydrazone ligands behave mostly as tridentate
ligands due to their short N-N bond length [2] for nickel complexes derived from
salicylaldehyde. This issue has been the focus of numerous studies in the past [3]. The
medicinal properties of their metal complexes depend upon the nature of the metal ions, with
the complexes having the ability to contain metal ions in different oxidation states [4]. N and
S atoms play a key role in the coordination of metal ions at the active sites of various metallo-
biomolecules [5]. Hydrazide-hydrazone derivatives play an important role in the development
of various biological activities, including [6] antifungal [7], anticancer [8], anti-diabetic [9],
antitubercular [10], antibacterial [11], antioxidant [12] and cytotoxic activity [13]. These
derivatives also play a key role in the modification of the electronic structures [14].
Especially, important biological activity has been observed in the complexes of metal ions
with an octahedral Cu(II) coordination preference [15]. The success of such treatment
indicated that the copper complexes have anticancer activity [16]. Various types of

interactions between small molecules and DNA have been identified, such as intercalation,
groove binding, electrostatic or ꢀ-ꢀ* ꢀinteractions [17]. The interaction of models to DNA–
drug systems is a subject of interest in the field of drug discovery [18]. Furthermore, the
binding of Schiff base metal complexes with the most abundant carrier proteins has been
employed to study drug-protein binding [19]. Bovine serum albumin (BSA) is one of the
most extensively studied bindings because of its structural homology with human serum
albumin (HAS) [20]. Thus, analyzing the syntheses of transition metal complexes which can
interact with protein has been the focus of many studies in recent years [21-22]. InhA, the
enoyl-acyl carrier protein reductases from Mycobacterium tuberculosis, is one of the key
enzymes involved in the mycobacterial fatty acid elongation cycle, and the crystal structure
of an InhA complex with one representative inhibitor reveals the binding mode of the
inhibitor within the InhA active site. Considering the problems in tuberculosis treatment,
developing new medications which address these critical issues is believed to be a health
priority which has attracted the attention of researchers. Molecular docking plays an
important role in the rational design of drugs to determine and predict the interaction of two
molecules [23-25]. In this paper, we explain the synthesis of complexes using a tridentate
ligand (Scheme 1). The complexes were characterized by FT-IR, UV-Vis, Raman, cyclic
voltammetry (CV) and single crystal X-ray studies. Also, the current article describes the
biological properties of the synthesized compounds via the customarily used methods for
studying drug-DNA and drug-BSA interactions, that include UV-visible spectroscopy,
fluorescence spectroscopy, cyclic voltammetry (CV) and circular dichroism (CD)
spectropolarimetry, and the in vitro antibacterial properties of the complexes were examined
against Gram-positive and Gram-negative bacteria. Furthermore, silico antituberculosis
properties were evaluated through a molecular docking simulation of InhA with the
complexes.

Scheme 1. Synthesis of complexes 1-3.
2
2
. Experimental
.1 Materials and methods
All solvents and chemicals were used as chemically pure. FT-IR spectra were obtained as
KBr plates using a Bruker spectrometer. UV-Vis spectra were obtained on a Shimadzu UV-
1
650PC spectrophotometer. Calf thymus DNA (CT-DNA), Tris-HCl (tris
hydroxymethylaminomethane-hydrochloride), methylene blue (MB), dimethyl sulfoxide
DMSO), dimethylformamide (DMF), bovine serum albumin (BSA) and all other chemicals
(
were analytical grade and obtained from Sigma-Aldrich. The Raman spectra were recorded
with SENTERRA Company: Bruker (Germany), and the analysis system SENTERRA was
equipped with powerful energy laser diodes, (these laser diodes exhale in the near infrared

region both visible and invisible laser radiation, according to the standard EN 60825-
1
/10.2003).
2
2
.2 Synthesis of H L
A 10 mL methanol solution of benzohydrazide (10 mmol) was added to a 10 ml methanol
solution of salicyilaldehyde (10 mmol) and the mixture was refluxed for 5 h. The resulting
light yellow precipitate was separated and filtered off, then washed with 4 ml of cooled
-1
methanol. Yield: 80%. M.P: 178 ºC. FT-IR, ꢁmax cm (KBr): 1622 (C=O), 1606 (C=N), 3269
-1
-1
(
N-H). UV-Vis, ꢂmax (nm) (ꢃ, M cm ) (DMSO): 392 (880), 306 (1010).
2
2
.3 Synthesis of thecomplexes
II
.3.1 Synthesis of [Cu
2
(L)
2
(py)
2
]·2ClO
4
(1)
To a stirring solution of Cu(ClO
4
)
2
·6H
2
O (0.5 mmol) in 10 mL methanol was added an
equimolar amount of H
2
L (0.5 mmol). The solution turned olive green immediately, due to
II
the formation of the [Cu (L)] complex. To this solution was added 0.5 ml pyridine and the
solution was stirred for 3 h to obtain a jade green solution. Single crystals suitable for X-ray
data collection were obtained by slow evaporation of the methanol solution after 7 days. The
crystals were filtered off, washed with a small amount of cold methanol and dried under
-1
vacuum. Yield: 71%. M.P: 390 ℃ . FT-IR, ꢁmax cm (KBr): 1556 (C=N), 1604 (C=O), 3188
-1
-1
(
N-H). UV-Vis, ꢂmax(nm)(ꢃ, M cm ) (DMSO): 546 (630), 330 (660).
II
2
2
.3.2 Synthesis of [Cu
(L)
2
(3-Mepy)
2
(ClO
4
)
2
] (2)
This complex was prepared by the same method as for complex 1, except that 0.5 ml 3-
methylpyridine was used instead of pyridine and ethanol was used as the solvent. Single
crystals of complex 2 suitable for X-ray crystallography were obtained by slow evaporation
of the ethanol solution after 5 days. The crystals were filtered off, washed with a small
-1
amount of cold ethanol and dried under vacuum. Yield: 69%. M.P: 402 ℃ . FT-IR, ꢁmax cm

-1
-1
(
(
KBr): 1598 (C=N), 1610 (C=O), 3064 (N-H), UV-Vis, ꢂmax (nm) (ꢃ, M cm ) (DMSO): 570
870), 328 (650).
II
.3.3 Synthesis of [Ni (L)(py)
2
3
]ClO
4
(3)
This complex was prepared by the same method as for complex 1, except that
Ni(ClO ·6H O was used instead of Cu(ClO ·6H O. Single crystals suitable for X-ray data
collection were obtained by slow evaporation of the methanol solution at room temperature
4
)
2
2
4
)
2
2
-1
after 5 days. Yield: 59%. M.P: 320 ℃ . FT-IR, ꢁmax cm (KBr): 1564 (C=N), 1602 (C=O),
-1
-1
3
191 (N-H). UV-Vis, ꢂmax (nm) (ꢃ, M cm ) (DMSO): 604 (890), 314 (930).
2
.4 X-ray crystallography
Single crystals suitable for X-ray diffraction were grown from methanol solution at room
temperature. Diffraction data were collected by the ꢄ-scan technique for 1 and 3 at room
temperature on an Agilent Technologies SuperNova four-circle diffractometer with an Atlas
CCD detector and mirror-monochromated CuK
ꢅ
radiation (ꢂ = 1.54178 Å), and for 2 at
1
20(1) K on an Agilent Technologies Xcalibur four-circle diffractometer with an Eos CCD
detector and graphite-monochromated MoK radiation (ꢂ = 0.71069 Å). The data were
ꢅ
corrected for Lorentz-polarization as well as for absorption effects [26]. Precise unit-cell
parameters were determined by a least-squares fit of the highest intensity reflections (12947
for 1, 6242 for 2, 8821 for 3) chosen from the whole experiment. The structures were solved
2
with SIR92 [27] and refined with the full-matrix least-squares procedure on F by SHELXL-
2
013 [28]. All non-hydrogen atoms were refined anisotropically; hydrogen atoms were
placed in idealized positions and refined with the ‘riding model’ using isotropic displacement
parameters set at 1.2 (1.5 for methyl groups) times Ueq of the appropriate carrier atoms. The
positions of those hydrogen atoms which cannot be reasonably placed by this procedure

(
water molecules) were calculated according to potential hydrogen bonds. The perchlorate
anions have been refined in two alternative positions in 1 and 3, with site occupation factors
refined at 58/42 and 72/28% in 1 and 3, respectively. Crystallographic data (excluding
structure factors) for the structural analysis has been deposited with the Cambridge
Crystallographic Data Centre, Nos. CCDC-1484884 (1), CCDC-1484885 (2) and CCDC-
1
484886 (3). Copies of this information may be obtained free of charge from: The Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK. Fax: +44(1223)336-033, e-mail:
deposit@ccdc.cam.ac.uk, or www: www.ccdc.cam.ac.uk.
Table 1. Crystal data, data collection and structure refinement.
Compound
1
32Cu
2
3
2
+
+
Formula
38
C H
2
N
6
-
O
4
C
40
H36Cl
2
Cu
2
N
6
O
12
·
C
29
H
26
N
5
NiO
4
2
·
-
2
(ClO
4
)
2H
2
O
ClO
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
962.67
monoclinic
C2/c
20.5256(5)
15.5068(4)
14.0472(3)
90
116.664(2)
90
1026.78
triclinic
P-1
634.71
monoclinic
P2
9.8311(2)
15.3478(3)
19.1823(3)
90
91.983(2)
90
2892.60(9)
4
1
9.9134(3)
12.7456(5)
18.7357(8)
108.403(4)
94.584(3)
108.383(3)
ꢅ (°)
β (º)
ꢆ (°)
V (Å )
3
3995.55(18)
4
2088.83(15)
2
Z
D
-
3
x
(g cm )
1.60
1.63
1.46
F(000)
µ (mm )
Θ range ( )
Reflections:
collected
1960
3.16
4.33 – 75.63
1052
1.22
3.32 – 26.64
1312
2.26
2.31 – 67.48
-1
0
15142
4037 (0.017)
3824
14462
7952 (0.017)
7029
14418
8494 (6.62%)
7489
unique (Rint
with I>2ꢇ(I)
)
R(F) [I>2ꢇ(I)]
wR(F ) [I>2ꢇ(I)]
0.050
0.151
0.027
0.065
0.062
0.162
2
R(F) [all data]
wR(F ) [all data]
Goodness of fit
max/min ∆ρ (e Å )
0.051
0.033
0.071
2
0.152
1.04
0.068
1.06
0.169
1.16
-3
0.61/-0.50
0.49/-0.38
0.94/-0.60

2
.5 Cyclic voltammetry
A Metrohm 757 VA computerace instrument was employed to obtain the cyclic
voltammograms in DMF solutions at room temperature (25 ºC) under a nitrogen atmosphere
using lithium perchlorate as the supporting electrolyte. A glass carbon working electrode, a
platinum auxiliary electrode and an Ag/AgCl reference electrode was used.
2
.6 Antimicrobial activity
In vitro antibacterial activities with different concentrations of H L and the complexes were
2
tested against bacterial species, including Escherichia coli (PTCC-1270), Klebsiella
pneumoniae (PTCC-1053) (Gram-Negative bacteria) and Staphylococcus aureus (PTCC-
1
112), Bacillus subtilis (PTCC-1254) (Gram-Positive bacteria). Kanamycin and Co-
trimoxazole were used as standard antibacterial agents. The minimal inhibitory concentration
MIC) was determined by the broth microdilution technique [29], which applied LB broth.
(
Sterile centrifuge tubes were arranged in rows of sets for each bacterial inoculum to cover the
different concentrations of the complexes. The MIC endpoint is the lowest concentration of
the compounds which produced inhibition of bacterial growth. The minimal bactericidal
concentration (MBC) is the lowest concentration of the antibiotic required to kill a particular
bacterium. In order to measure the MBC, 100 µL volumes of all clear tubes from a dilution
MIC test were spread on Mueller Hinton agar plates and incubated at 37 °C overnight.
Solutions of the compounds in DMSO were used for the studies and the experiments were
measured after 24 h [30].
2
.7 Protein binding studies
All CD measurements were obtained employing an Aviv 215 (Aviv Instrument, Inc., Lake
Wood, NJ, USA) spectropolarimeter by keeping the concentration of bovine serum albumin
-4
-3
constant ([BSA = 0.5×10 mol dm ]), in the presence of the synthesized compounds with

-4
-3
concentrations of 0.5 and 1ꢀ×10 mol dm . A stock solution of BSA was prepared by
dissolving a suitable amount of BSA in aqueous solution containing phosphate buffer (0.01
M, pH 7.4) at ambient temperature. All spectra were recorded in the far-UV range (200-260
nm).
2
2
.8 DNA binding experiments
.8.1 Preparation of stock solution
The CT-DNA stock solution was prepared by dissolving an appropriate amount of CT-DNA
in Tris-HCl (tris hydroxymethylaminomethane-hydrochloride) buffer (0.01 M, pH 7.4) at
ambient temperature. A stock solution of methylene blue was prepared by dissolving its
crystals in deionized double-distilled water. The solutions of the synthesized compounds
were prepared by dissolving the synthesized compounds in DMSO.
2
.8.2 Absorbance spectroscopy
A UV-Vis spectroscopic study was performed using a UV-visible spectrophotometer (T-60,
-5
PG Instrument UK). In addition, a fixed amount of CT-DNA (5 ×10 M) was titrated with
increasing amounts of solutions of the synthesized compounds in the wavelength range 240-
3
00 nm.
2
.8.3 Emission spectroscopy
Fluorescence spectra were achieved using a JASCO FP-750 spectrofluorimeter (Tokyo,
Japan) equipped with a 150 W Xenon lamp, using a 1.0 cm quartz cuvette. The excitation
wavelength was set at 630 nm. The fluorescence measurements were monitored by keeping
the concentration of CT-DNA and MB (methylene blue) constant, while varying the
-5
concentration of the synthesized compounds, [CT-DNA] = [MB] = 5 ×10 M, in the
wavelength range 650-790 nm.
2
.8.4 Circular dichroism spectropolarimetry

-
5
The CD spectra of of DNA (5× 10 M) were recorded in the far-UV range (230-290 nm) in
the presence of increasing amounts of H L and complexes 1-3, with concentrations of 7 and
2
-6
9
ꢀ×10 M.
2
.8.5 Electrochemical studies
Cyclic voltammetry measurements were recorded with a Potentiostat/Galvanostat (Model
63A, EG&G, USA) under a nitrogen atmosphere and carried out for [CT-DNA] =
2
-5
[
complexes] = 5 ×10 M, by the electrodes mentioned in Section 2.5.
2
.9 Molecular docking simulation studies
All the software applied for this analysis are freely available for academic use. The Protein
Data Bank (www.rcsb.org), as a universal repository for the processing and distribution of 3D
biological macromolecular structure data, was used for downloading InhA (PDB code:
2
NSD) [31]. The docking simulations were performed using the Hex docking software [32].
The structures were optimized with the Argus lab program after deleting the ClO
crystallographic data [33].
4
anion from
3
3
. Result and Discussion
.1 Synthesis and characterization
The nickel and copper complexes have been prepared by the direct reaction of the tridentate
Schiff base ligand with Ni(II) perchlorate and Cu(II) perchlorate, respectively, in the presence
of pyridine and 3-methylpyridine .The crystals were filtered off, washed with a small amount
of cold methanol and dried under vacuum.
3
.2 IR and Raman spectra

The IR spectra of the Schiff base ligand (H
most of the main peaks. The IR spectrum of H
complexes (1-3). The IR spectrum of H L reveals a band in the 3200-3600 cm region,
2
L) and its complexes (1-3) were recorded for
2
L was compared with the spectra of
-1
2
assigned to the ꢈ(OH) group. The disappearance of this band in the spectra of complexes 1-3
indicates the deprotonation of the hydroxyl group with the coordination of the phenolic OH
-
1
group. The strong band observed at 1606 cm in the free Schiff base ligand is allocated to the
azomethine group vibration. With the coordination of the imine nitrogen atom to the metal
-1
ions, this band is shifted towards lower frequencies by 1556, 1598 and 1564 cm in
complexes 1, 2 and 3, respectively. Two new bands appear in the low frequency ranges 695-
-1
7
06 and 620-699 cm which have been assigned to the ꢈ(M-O) and ꢈ(M-N) vibrations
respectively. A comparison of the IR spectra of the Schiff base ligand and its metal
complexes indicates that H L is coordinated with the metal atom through three sites,
suggesting that H L acts as a tridentate ligand. Raman spectroscopy is a spectroscopic
2
2
technique used in condensed matter physics and chemistry to study vibrational, rotational and
other low-frequency modes in a system. The C=N stretching vibrations in the Raman spectra
-1
of 1, 2 and 3 were observed at 1599, 1571 and 1565 cm , respectively, and the C=O
stretching vibrations in the Raman spectra of 1, 2 and 3 were observed at 1604, 1606 and
-
1
-1
1
603 cm , respectively. The Raman bands from 1000 to 1200 cm were assigned to the C-C
-1
and C-O stretching vibrations and the peaks between 1200 and 1300 cm are due to the C–H
-
1
bending in the aromatic ring. Also, the peaks between 1400 and 1500 cm probably reflect
the CH bending vibrations [34].
.3 Electronic spectra
The UV-Vis absorption spectra of H
2
3
-4
2
L and its complexes (1× 10 M) in DMSO were
recorded at room temperature. Electronic absorption spectra are often helpful in evaluating
the results obtained through other methods of structural investigation. The electronic

absorption spectrum of H
→ ꢁ* transitions of the benzene ring, and a band at 392 nm, corresponding to ꢂ → ꢁ*,
which upon coordination of the ligand disappeared from the UV-Vis spectra of complexes 1-
. Also, another band at 306 nm was assigned to theꢀꢁ → ꢁ* transition of the azomethine
2
L consists of a relatively intense band at 254 nm, assigned to the
ꢁ
3
group. This band shifted in the corresponding complexes 1-3 and appeared in the range 313
to 331 nm, indicating the coordination of the C=N group to the metal ions. The d-d
transitions in the three complexes are assessed at about 546 nm for 1, 570 nm for 2 and 604
nm for 3.
3
.4 X-ray studies
Perspective views of complexes 1-3 are presented in Fig. 1(a-c), and some of bond distances
and bond angles around the metal centers are presented in Table 2. In the crystal structures of
2
and 3 there are two symmetry-independent complex molecules; in fact, in 2 there are two
independent halves of the dimer complexes (across two different inversion centers). In the
copper complexes 1 and 2 there are four shorter Cu-N and Cu-O bonds and one (1) or two (2)
significantly longer Cu-O contacts, which should probably be regarded as bonding contacts.
Taking them into account, both complexes are two centered and symmetrical (C
2
for 1, C
i
for
2
). In 1, the Cu cation is five coordinated by two nitrogen and two oxygen atoms from L (one
of the oxygen atoms, O1, is in a bridging position), and by one nitrogen atom of the
coordinated pyridine molecule. The geometry around the Cu atom is close to square
pyramidal. In complex 2, the perchlorate anion is additionally coordinated (by a weaker
contact), and one of its oxygen atoms takes the sixth position which completes the octahedral
geometry. In two symmetry-independent molecules of Ni complex 3, the metal cations are six
coordinated by an N
2
O system of the L molecule. With the three pyridine molecules, the
geometry is close to an ideal octahedral one. In the crystal structures, hydrogen bonds (Table

2
) together with Coulombic interactions and van der Waals forces determine the packing of
the structural elements.
“
Here Figure 1”
Table 2. Relevant geometrical features of complexes 1-3 (Å, °). The two (c.n. 5) or three
c.n. 6) largest angles around the metal center are listed; A, B, C denote the mean planes of
the terminal phenyl rings and central approximately planar linker.
(
1
(Cu)
2A (Cu)
1.9112(13)
1.9469(15)
1.9887(13)
1.9862(15)
2.5764(13)
2.5833(14)
2B (Cu)
1.9002(13)
1.9443(15)
1.9683(13)
1.9907(15)
2.7323(14)
2.6111(16)
3A (Ni)
1.987(5)
2.016(6)
2.131(6)
2.136(7)
3B (Ni)
2.001(6)
2.019(7)
2.108(5)
2.119(7)
M-O1A
M-N8A
M-O10A
M-N17A
1.917(2)
1.932(2)
1.9855(19)
1.974(2)
2.586(2)
i
M-O1A
M-O (ClO )
4
M-N (pyridine)
2.136(7)
2.119(7)
2.152(7)
167.8(2)
172.0(3)
176.6(3)
2.155(7)
1
1
71.00(8)
69.48(10)
173.34(4)
171.89(6)
173.47(4)
173.26(6)
169.99(6)
177.1(3)
174.4(3)
168.08(19)
1
69.83(5)
A/B
B/C
A/C
5.52(15)
17.37(18)
22.35(17)
9.8(3)
10.7(3)
4.18(12)
7.9(2)
14.3(2)
6.79(11)
8.3(8)
25.0(6)
21.2(4)
7.9(9)
19.4(8)
23.9(5)
3
.5 Electrochemical studies
The cyclic voltammetric spectra for complexes 1-3 were recorded in DMF solution at
ambient temperature (300 K) with glass carbon, platinum and Ag/AgCl electrodes as
working, auxiliary and reference electrodes, respectively. Lithium perchlorate was also used
as a supporting electrolyte (2 M). For a comparison of the cyclic voltammograms of 1, 2 and
3
see Figs. 2-4. The voltammograms of 1 and 2 are somewhat similar, while 3 is
conspicuously different. During the cathodic scan, 1 and 2 were observed to have different
reductive responses, at -0.44 and -0.57 V for 1, -0.58 and -0.75 V for 2, while only a single
reductive response was observed at -0.87 V for 3. Similarly, during the anodic scan, oxidative
responses were observed at 0.23 and 0.31 V for 1, 0.60 and 0.39 V for 2, whilst there was not

a clear oxidative peak for 3. Scans for 1 and 2 have been assigned to the reduction of the
Cu(II) ꢉ Cu(I) centers and to the oxidation of the Cu(I) ꢉ Cu(II) centers in the complexes,
and the scan for 3 has been assigned to the reduction of the Ni(II) ꢉ Ni(I) center in the
complex. The first reduction potential for Cu(II/I) and Ni(II) ꢉ Ni(I) is irreversible, with the
concomitant release of the axial (amine) ligands.
“
“
“
Here Figure 2”
Here Figure 3”
Here Figure 4”
3
.6 Antibacterial activity
Recently, a series of hydrazone derivatives were reported to have antibacterial activity
against Gram-positive and Gram-negative bacteria. Several related compounds showed good
antibacterial activity against Gram positive bacteria. Their activities were highly dependent
on the structure of the different compounds involved [35-37]. In this report, the ligand and its
complexes were screened separately for their antibacterial activity against Escherichia coli,
Klebsiella pneumoniae (Gram-Negative) and Staphylococcus aureus, Bacillus subtilis (Gram-
Positive). These compounds were dissolved in DMSO. The minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC) values of H
complexes 1-3 are listed in Table 3 and Fig. 5(a,b). According to Table 3, it is significant that
the metal complexes exhibited a greater inhibitory effect compared to H L. It seems that the
2
L and
2
metal complexes cause disorder in the membrane structure, destroying the electrochemical
slope between cytosol and the external environment due to the interaction with the membrane
and resulting in physiologic death of bacteria. The polarity of the metal ion is considerably
decreased by chelation because of the ꢀ-electron delocalization over the whole chelate and
also due to partial sharing of its positive charge with the donor group. Furthermore, the
reduction in polarity increases the lipophilic nature of the chelates, thus an interaction

Table 3. MIC and MBC of the synthesized compounds against growth of bacteria (×
ꢃꢄ
1
0 Mꢀ).
Gram-Positive Bacteria
Gram-Negative Bacteria
K. pneumoniae E. coli
Compound
B. subtilis
S. aureus
MBC
416
MIC
104
MBC MIC
MBC
416
MIC
416
MBC
416
MIC
416
H
1
2
L
416
8.2
208
4.0
8.2
2.0
65.6
126.5
93.6
32.8
63.2
93.6
131.2
63.2
93.6
65.6
63.2
48.8
2
15.8
46.8
7.9
15.8
93.6
15.8
46.8
3
23.4
between the lipid and the metal ion is favored, which can be explained on the basis of
chelation theory [38].
The results indicated that the synthesized compounds have better activity towards Gram-
positive bacteria than towards Gram-negative bacteria. The reason for this may probably be
due to the difference of the cell membrane structure, where the outer membrane of Gram-
negative bacteria can reduce the damage from the synthesized compounds. This means that
the external membrane of Gram-negative bacteria may play a preventive role for the
antibacterial effect of the above-mentioned compounds. The strongest growth-inhibitory
effect (MIC) is related to complex 1, which has been observed in B. subtilis, and the strongest
effect of MBC is related to complex 1 on B. subtilis and S. aureus bacteria. Also, the results
suggest that replacing copper with nickel improves the antibacterial property in Gram-
positive bacteria. For compounds 1 and 2, the substitution of 3-methylpyridine with pyridine
-
and also linking the ClO
significantly.
4
anion to the copper center has weakened the antibacterial property
“
Here Figure 5”
3
3
.7 BSA binding experiment
.7.1 Circular dichroism spectropolarimetry

Circular dichroism (CD) spectropolarimetry analysis gives us useful information about the
secondary structure conformational changes induced by the binding of the synthesized
compounds to BSA. Serum albumin is considered as a drug transporter protein. This method
is very suitable because BSA has a high percent of the ꢅ-helical structure which shows a
specific CD signal in the far-UV region. CD spectra containing a fixed concentration of BSA
-4
-3
(
0.5×10 mol dm ) and varying concentrations of H
2
L and complexes 1-3 are shown in Fig.
6
. As expected, for a protein that is mainly ꢅ-helical, the CD spectrum of BSA shows a
negative ellipticity at 209 and 220 nm (Fig. 6, curves a). The intensity of this double
minimum reflects the amount of helicity of BSA when the conformation of the BSA molecule
is changed significantly in the presence of the synthesized compounds [39-41].
“
Here Figure 6”
L and complexes 1-3 causes a decrease in the band intensity with a very
weak blue shift. Similar spectral features were observed for the interaction of both the
complexes and H L with BSA. The differences indicate that the interaction of the Schiff base
ligand and the complexes with BSA changes the secondary structure of BSA. Fractional
contents of the secondary structure of BSA (f , f L and
complexes 1-3 are calculated using the CDNN program [42] and the results are shown in
Table 4. Our CD results show that the addition of H L and complexes 1-3 to BSA solution
caused the reduction of the ꢅ-helix fraction (fꢅ) and the fraction of turn (fturn). Also, our CD
results show that the addition of H L and complexes 1-3 to BSA increased the fraction of fꢊ-
sheet (f ), and random coli (frandom). These studies were carried out to ensure from the
The addition of H
2
2
ꢅ
ꢊ
, fturn, and frandom) with and without H
2
2
2
ꢊ
capability of the interaction between these compounds and serum albumin, the consequent
possibility of their transition in the blood, and the interaction between these compounds and
serum albumin was tested using the spectral changes in the far-UV region and by the changes
in the secondary structure. The binding constants (K) of the synthesized compounds with

BSA are listed in Table 5, which were calculated based on Kim and Baldwin’s method [43]
under equilibrium conditions and using the following equation:
ƒ
U
= (ꢋ
F
– ꢋ) / (ꢋ
F
U
– ꢋ )
(1)
K = ƒ
U
/ (1 - ƒ
U
) = ƒ
U
/ ƒ
F
= (ꢋ
F
– ꢋ) / (ꢋ – ꢋ
U
)
(2)
where ƒ
conformations, respectively; ꢋ
unfolded states, respectively [44].
F
and ƒ
U
represent the fraction of BSA present in the folded and unfolded
F
and ꢋ represent the values of ellipticity of the folded and
U
-4
-3
Table 4. Fractional contents of the secondary structure of BSA (0.5×10 mol dm ) with and
without H
2
L and complexes 1-3.
Concentration ratios of BSA
Compound
f
ꢅ
f
ꢊ
f
turn
f
random
to H
2
L and complexes 1-3
1
:0
0.416
0.133
0.129
0.152
0.324
0.327
0.169
0.164
0.164
0.260
0.377
0.377
H
2
L
1:1
1
:2
:0
1
0.416
0.194
0.153
0.416
0.152
0.270
0.309
0.152
0.169
0.169
0.168
0.169
0.260
0.365
0.367
0.260
1
2
1:1
1
:2
:0
1
1
:1
:2
0.205
0.162
0.260
0.299
0.169
0.168
0.364
0.367
1
1:0
0.416
0.152
0.169
0.260
3
1
:1
:2
0.188
0.149
0.275
0.315
0.169
0.167
0.367
0.370
1
Table 5. Binding constants for the interaction of the compounds with BSA
-1
Compound
Binding constant (M )
3.0 × 10ꢅ
2
H L
1
2
1.5 × 10ꢆ
1.4 × 10ꢆ

3
1.2 × 10ꢆ
According to Table 5, these values suggest that the binding of H L with BSA is weaker than
2
with the complexes. Moreover, complex 1 exhibits a comparatively stronger binding affinity
to BSA. Thus, it is important that their interaction with BSA depends on the nature of the
metal ion and the compound’s structure, which is in good agreement with the previously
known metal complexes of hydrazide derivatives [45-46].
3
3
.8 DNA binding experiments
.8.1 Absorption characteristics of DNA-drug
Fig. 7 shows the absorption spectra of DNA with or without the ligand and complexes 1-3 at
various concentrations. Here, the DNA-binding studies with H L and complexes 1-3 suggest
2
their potential to interact with DNA. This shows that while adding the ligand and the
complexes to a solution of DNA, the DNA gets adsorbed on the surface of the drug and is
involved in the formation of a ground state complex of the type DNA…drug. As can be seen
in Fig 7, a hyperchromism effect is observed when the synthesized compounds are added to
the DNA. This effect can be attributed to external contact (surface binding) with the duplex
DNA which damages the DNA double-helix structure. The double stranded DNA molecules
revealed a typical hyperchromic effect during the transition to partial or completely single
stranded DNA, in the other words, this effect is evident from the change in absorbance when
the DNA undergoes a conformational transition from double-stranded to single-stranded
structure in response to chemical perturbation. Also, the spectral changes of DNA observed
during the addition of H
2
L and the complexes, without any change in the location of the peak
(
258 nm), revealed that the interaction may occur via a non-intercalative binding mode,
exclusively to the periphery of the double helix [47-49].The extent of the hyperchromism is
indicative of the non-intercalative binding mode, revealing that the binding of the synthesized

compounds with DNA is also supported by electrostatic, van der Waals and H-bonding
interactions [50]. The equilibrium for the formation of a compound between DNA with H
2
L
or the complexes is given by Eq. (4), where Kapp represents the apparent association constant:
Drug: Ligand or complex
DNA + drug ⇌ DNA…drug
(3)
[
ꢇꢈꢉ…ꢊꢋꢌꢍ]
K
app = [
(4)
ꢇꢈꢉ]ꢀ.ꢀꢀ[ꢊꢋꢌꢍ]
In this case, Kapp is obtained based on Benesi and Hildebrand’s method [51].
obs = (1- ꢅ)C ꢍ (5)
where Aobs is the observed absorbance of the solution, containing various concentrations of
drug at 258 nm, ꢅ is the degree of association between DNA and drug, ꢌDNA and ꢌ are the
molar extinction coefficients at the defined wavelength (ꢂ = 258 nm) for DNA and the
A
0
ꢌ
0 c
DNAꢍ + ꢅC ꢌ
c
formed compound, respectively, 'l' is the optical path length and C
0
0
is the primary
and A are the
concentration of DNA. Eq. (5) can be expressed as Eq. (6), where A
c
absorbances of DNA and the compound at 258 nm, respectively, with a concentration of C
+ ꢅA (6)
ꢅ can be equated to (Kapp[drug])/(1 + Kapp[drug]), thus Eq. (4) can be changed to Eq. (7) [52]:
0
:
A
obs = (1 – ꢅ)A
0
c
ꢎ
ꢎ
ꢎ
ꢎ
=
+
.
ꢒ)ꢀ
ꢀꢀꢀ(7)
ꢉꢏꢐꢑꢃꢉꢒ
ꢉꢓꢃꢉꢒ
ꢔꢕꢖꢖ(ꢉ ꢃꢉ
ꢀ[ꢊꢋꢌꢍ]
ꢓ
Based on the enhancement of the absorbance at 258 nm due to absorption of the DNA-drug
complex, we would expect a linear relationship between 1/(Aobs – A ) and the reciprocal
concentration of drug, with a slope equal to 1/Kapp(A – A ) and an intercept equal to 1/(A
) [Fig. 7, insets]. The values of the determined apparent association constants (Kapp) are
0
c
0
c
-
A
0
summarized in Table 6 and compared with the values of the binding constants of previously
known metal complexes of hydrazide derivatives [53-54]. The binding constant is related to
the compound’s structure. Here, according to the structural features, spectral changes and
binding constant, the reaction between the synthesized compounds with DNA is a weak

reaction. Stable metal complexes are extremely valuable as probes for biological systems,
which can be ascribed to the effect of the metal ion in binding the drug with DNA [55]. Thus,
according to binding data, the metal complexes have a stronger interaction with DNA than
H
2
L; also using copper center instead of a nickel center causes a stronger interaction with
DNA. Also for compounds 1 and 2, the substitution of 3-methylpyridine instead of pyridine
and also linking the ClO anion to the copper center weakens the binding with DNA. In
general, the DNA-binding affinities of H L and complexes 1-3 decrease in the order 1 > 2 >
> H L.
4
2
3
2
Table 6. Binding constants for the interaction of the compounds with DNA
-
1
2
Compound
Binding constant (M )
1.6 × 10ꢅ
R
H
2
L
0.98
0.98
1
16.6 × 10ꢅ
2
3
14.2× 10ꢅ
10× 10ꢅ
0.97
0.99
“
Here Figure 7”
3
.8.2 Fluorescence studies
Fluorescence spectra are used to study the interaction between metal complexes and DNA.
The fluorescence of certain polycyclic cationic dyes is quenched upon intercalation between
base pairs in polynucleotides such as DNA. Fluorescent compounds such as methylene blue
(
MB) and ethidium bromide are normally used to probe the DNA structure in drug-DNA
interactions. Methylene blue is a tricyclic [56-57] cationic dye that binds to calf thymus DNA
CT-DNA). The emission spectra of the MB–DNA solution in the presence of increasing
concentrations of H L and complexes 1-3 is shown in Fig. 8. Upon binding to DNA, the
fluorescence from MB is efficiently quenched by the DNA bases due to rapid electron
(
2

abstraction from an adjacent nucleic acid base by the excited singlet state of the dye [58-59].
If the interaction between the ligand and metal complexes 1-3 with DNA causes MB-DNA
separation (separation of MB from DNA), a significant increase in the MB emission is seen.
Here, there is no significant increase in the fluorescence intensity of the probe molecule
during the addition of H
2
L and complexes 1-3, indicating that the MB molecules are not
L and complexes 1-3 and considering that
released from the DNA helix after addition of H
2
the interaction between MB and DNA is intercalation. Also, the significant lack of change in
the emission spectra suggests a non-weaving interaction of the ligand and metal complexes
with DNA. In another words, the interaction between MB and DNA is intercalating and the
interaction between the ligand and metal complexes with DNA is non-intercalating [49,60].
“
Here Figure 8”
3
.8.3 Circular dichroism spectropolarimetry
The changes in the CD signals of DNA observed on interaction with drugs may often be
assigned to the corresponding changes in the DNA structure. CD spectra were measured by
keeping the concentration of CT-DNA constant while varying the concentration of drug. The
characteristic B-form of free CT-DNA CD spectra shows a positive band at 276 nm due to
base stacking and a negative band at 244 nm due to right handed helicity [61-63]. The
changes in the DNA structure can be monitored by using the CD spectra [64]. Fig. 9 shows
that the addition of H L or complexes 1-3 to DNA decreases the intensities of both the
2
negative and positive bands significantly (shifting to zero levels). This shows the structure of
CT-DNA is affected by the addition of the drug and the change is much higher for higher
concentrations of the drug. The interaction with DNA could induce certain conformational
changes, such as the conversion from a more B-like to a more C-like structure within the

molecule. These results also confirm the non-intercalative mode of binding of CT-DNA with
the drug [49,65].
“
Here Figure 9”
3
. 8.4 Electrochemical studies
The cyclic voltammograms (CV) of the complexes in the absence and presence of the CT-
DNA were recorded in the range −1.2 to 1.5 V. It can be seen from Figs. 10-12, in the
absence of DNA (curves a), the complexes showed different reductive responses at -0.54 and
-
0.30 V for 1, -0.72 and -0.42 V for 2, corresponding to the Cu(II)/Cu(I) couple, and anodic
peak potentials at 0.29 and 0.61 V for 1, 0.47 and 0.76 V for 2, which have been assigned to
the oxidation of the Cu(I) ꢉ Cu(II) centers in the complexes. Also, only a single reductive
response is observed at -0.81 V for 3, Ni(II)/Ni(I), and there no clear oxidative peak. In the
presence of CT-DNA (curves b), the Epa and Epc peaks potentials shifted to more negative
values and also in the presence of DNA the voltammetric peak currents increased. These
effects describe the interaction of the complexes with CT-DNA via a non-intercalative mode
of binding [65], because if a molecule intercalated into the DNA molecule, the peak
potentials show a shift to a more positive potential; moreover, the peak currents decrease. In
general, an increment in the peak currents of complexes in the presence of CT-DNA could be
related to the interaction between the complexes and CT-DNA [66-68].
“
“
“
Here Figure 10”
Here Figure 11”
Here Figure 12”

3
. 9 Molecular docking simulation studies
The binding of drugs with 2snd plays an important role in the study of the bioavailability,
efficacy, transport and designing of the drugs against mycobacterium tuberculosis [69].
Docking is conducted by a procedure involving the multiple conformer shape-matching
alignment of a molecule to a cavity, followed by energy minimization on both the complexes
and the InhA residues at the binding region. Hex is an interactive program which lets a user
load a pair of proteins, view them in a variety of 3D graphical modes, perform docking
calculations and then visualize the results. A pair of InhA structures in “PDB” format
(
http://www.pdb.org) were docked to the complexes of 1, 2 and 3 using the default of Hex
and default evolutionary parameters. The results of the docking of 2nsd with the complexes
are summarized in Table 7 and are shown in Fig. 13. According to these, the InhA docking
energy values are obtained as -397.68, -385.11 and -349.54 kcal/mol for 1, 2 and 3,
respectively. It is clear that compound 1 shows a greater binding energy value (-397.68
kcal/mol) and thus seems the most appropriate potential drug in this case.
Table. 7 Hex results for the docking of enoyl-acyl carrier protein (2nsd) with complexes 1-3.
Compound
Total energy
Distance between complex Fast Fourier Transform
(
kcal/mol)
-397.68
and receptor (R, Å)
16.0
(FFT, N)
25
1
2
-385.11
-349.54
20.8
20.8
25
25
3
“
Here Figure 13”
4
. Conclusion
In this study the metal complexes of a tridentate Schiff base ligand have been synthesized
and characterized by FT-IR, UV–Vis and Raman spectroscopy, and cyclic voltammetry
(
CV). The crystal structures of the complexes were determined by X-ray crystallography.

The antibacterial activities of the synthesized compounds against bacterial strains were also
examined. Furthermore, the present article focuses on analyzing ligand-DNA, complex-
DNA, ligand-BSA and complex-BSA interactions by applying various optical methods,
including UV-visible spectroscopy, fluorescence spectroscopy, cyclic voltammetry (CV) and
circular dichroism (CD) spectropolarimetry. The results of the binding of the ligand and the
complexes to CT- DNA resulted in a hyperchromic shift, showing the non-intercalative
mode. Also, quantitative analysis of far UV-CD spectra showed significant alterations of the
BSA secondary structure in the presence of the ligand and the complexes in aqueous
solution, with reduction of the α-helices content. Thus, it can be concluded that these
complexes act as antibacterial agents and DNA-binding components and hence can be
considered as potential pharmaceutical bioactive compounds. Also, the findings related to
BSA-binding may reveal their capability to participate in transport and elimination processes
in the body and therefore, it is necessary to use this information in drug design. Finally,
molecular docking simulation studies were conducted and the results indicated that complex
1
is an appropriate potential candidate drug for the binding the InhA target in mycobacterium
tuberculosis.
. Acknowledgements
5
We gratefully acknowledge the Faculty of Chemistry at Semnan University and the
University of Mohaghegh Ardabili for supporting this work.
6
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